Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.     

Release of copper from yeast copper-thionein after S-alkylation of copper-thiolate clusters.

Our knowledge on the release of copper from Cu-thionein in biological systems is limited. Other than oxidative cleavage or direct transfer, the possibility of an alkylation mechanism seemed attractive. Iodoacetamide and methyl methanesulphonate were successfully employed to alkylate the Cu-thiolate sulphur atom of homogeneous Cu(I)-thionein from yeast. The alkylation caused a weakening of the Cu-S bonding, which led to the release of copper. After equilibrium dialysis a proportion of the released copper was found in the dialysis buffer. When iodoacetamide was used carboxymethylcysteine was detected in the protein hydrolysate. A 10-fold molar excess over cysteine was sufficient for complete alkylation, which could be conveniently monitored by c.d. at 328 and 359 nm. The reaction proceeded under both aerobic and anaerobic conditions. E.p.r. measurements of Cu2+ revealed unequivocally the complete cleavage of the Cu-thiolate bonding in less than 5 h. It is possible that this mode of copper release might be of relevance to the molecular transport of this biochemically important transition metal.     

Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Mugharet el'Aliya: Affinities of an enigmatic north African Aterian maxillary fragment

Objectives

This study uses a virtual framework to examine the left maxillary fragment of the juvenile fossil from Mugharet el'Aliya, Morocco, found in association with an Aterian lithic industry. Previously, this fossil had been ascribed to modern humans or the Neanderthal lineage based on its “archaic”/“Neanderthal-like” features and apparent large size. Here, we conducted a novel 3D shape comparative analysis of the maxillary fragment to clarify its taxonomic affinities with regard to its size and ontogeny.

Materials and Methods

Eighty Computed Tomography and surface scans representing ontogenetic samples of Homo sapiens and Homo neanderthalensis were used to capture species-specific differences. The toolkit of geometric morphometrics in combination with surface registration and an elastic iterative closest point algorithm were used to create a dataset of meshes with an identical number of corresponding vertices for the maxillae. Multivariate statistics were applied to Procrustes superimposed coordinates derived from the vertices of this dataset.

Results

Our analysis showed affinities of the Mugharet el'Aliya individual with our H. sapiens sample, especially with a subadult individual from Qafzeh. No size-independent affinities with Neanderthals of comparable dental age could be identified.

Discussion

Our results add to the evidence connecting fossils from western Asia, especially Qafzeh and Skhul, and the North African Aterian. Furthermore, Mugharet el'Aliya adds to our knowledge of the ontogenetic development of adult morphology that is frequently used to characterize hominin groups, for example, Neanderthals and modern humans.